Tuberculosis, caused by the etiological agent Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide from a curable infectious agent and is becoming a major concern due to the spread of drug resistant Mtb strains. Notably, Mtb can persist in a dormant, drug resistant state, sometimes reactivating to cause TB decades after the primary infection. Currently, there is strong interest in exploiting oxidative phosphorylation (OXPHOS) as a metabolic target for new anti-TB drugs and drug combinations. In this regard, there are several antimycobacterial drugs that target the Mtb electron transport chain (ETC), including bedaquiline (the first new TB drug in ~40 years), Q203, clofazimine, and phenothiazines. However, how inhibition of respiratory complexes in the ETC leads to effective killing has yet to be established. We believe there is a critical gap in our understanding of how OXPHOS communicates with central carbon catabolism in response to changing environmental fuel sources to survive the host immune response and anti-TB drug therapy. Our long-term goal is to define the bioenergetic mechanisms that enable Mtb to survive within the host in a dormant, drug resistant state. In this proposal, our central hypothesis is that the interplay between the Mtb ETC and central carbon catabolism prevents effective killing by anti-TB drugs. To test this hypothesis, we have established a series of specific aims to determine how OXPHOS-generated ATP modulates central carbon catabolism and succinate excretion to maintain metabolic homeostasis, examine the mechanisms whereby simultaneous inhibition of OXPHOS and glycolysis kills Mtb, and test the hypothesis that bioenergetic homeostasis in clinical strains of Mtb contributes to drug tolerance. We will make use of a novel technology termed extracellular flux (XF) analysis that we have adapted for studying Mtb bioenergetics in real time. This technology will be complemented by 13C stable isotope analyses using liquid chromatography mass spectrometry This contribution is significant, because it has the potential to identify a new paradigm that will lead to a detailed mechanistic understanding of how the Mtb ETC communicates with central carbon catabolism, and how disruption of this process could be exploited to sterilize Mtb. This proposal is innovative in our opinion, because the newly adapted technology that is supported by metabolomics, distinguish itself from conventional approaches for studying energy metabolism in pathogenic microbes.